Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors

Chan, Katie; Robinson, Margaret; Yang, Huiling; Cornew, Sophia; Delaney, William E.

doi:10.1016/j.antiviral.2013.01.005

Cited by 7 publications

(3 citation statements)

References 24 publications

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“…Previous studies suggested that in vitro adaptive mutations enhance the production of infectious virus[ 13 - 17 , 20 , 21 , 30 - 34 ]. A high mutation rate in HCV RNA genome is a challenge for successful HCV treatment and vaccine research, although a method to obtain a robust clonal culture of HCV has been unavailable[ 13 ].…”

Section: Discussionmentioning

confidence: 99%

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

Wang

Liu

et al. 2018

WJG

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AIMTo explore hepatitis C virus (HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODSA series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and cell lysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets (LDs) with NS5A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5A was analyzed further.RESULTSThe plasmids named JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/NS5A, and mJFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units (FFUs)/mL. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were critical adaptive mutations. The effect of NS5A (C2274R, I2340T, and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A.CONCLUSIONIn this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.

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Section: Discussionmentioning

confidence: 99%

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

Wang

Liu

et al. 2018

WJG

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“…Reporter genes were either fused to viral proteins (i.e. NS5A) (Gottwein et al, 2011a(Gottwein et al, , 2011b or added as additional proteins (Andrus et al, 2011;Liu et al, 2012Liu et al, , 2014aChan et al, 2013;Horwitz et al, 2013), that are cleaved off the viral polyprotein by the NS3/4A protease (i.e. by duplicating the NS5A-B cleavage site) (Fig.…”

Section: Model Systems To Study the Complete Hcv Life Cyclementioning

confidence: 99%

Advances in experimental systems to study hepatitis C virus in vitro and in vivo

Catanese

Dorner

2015

Virology

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Hepatitis C virus (HCV) represents a global health concern affecting over 185 million people worldwide. Chronic HCV infection causes liver fibrosis and cirrhosis and is the leading indication for liver transplantation. Recent advances in the field of direct-acting antiviral drugs (DAAs) promise a cure for HCV in over 90% of cases that will get access to these expensive treatments. Nevertheless, the lack of a protective vaccine and likely emergence of drug-resistant viral variants call for further studies of HCV biology. With chimpanzees being for a long time the only non-human in vivo model of HCV infection, strong efforts were put into establishing in vitro experimental systems. The initial models only enabled to study specific aspects of the HCV life cycle, such as viral replication with the subgenomic replicon and entry using HCV pseudotyped particles (HCVpp). Subsequent development of protocols to grow infectious HCV particles in cell-culture (HCVcc) ignited investigations on the full cycle of HCV infection and the virus-host interactions required for virus propagation. More recently, small animal models permissive to HCV were generated that allowed in vivo testing of novel antiviral therapies as well as vaccine candidates. This review provides an overview of the currently available in vitro and in vivo experimental systems to study HCV biology. Particular emphasis is given to how these model systems furthered our understanding of virus-host interactions, viral pathogenesis and immunological responses to HCV infection, as well as drug and vaccine development.

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“…For this reason, bioluminescence is mainly used for in vitro measurements; macroscopic and microscopic observations are less common. Luciferase reporter viruses have been designed mostly for animal and human viruses, for example, hepatitis C virus, [6], herpes simplex virus 1 [7], HIV-1 [8], or dengue virus [9]. Examples of luciferase-tagged plant viruses are scarce.…”

Section: Introductionmentioning

confidence: 99%

Bioluminescence Production by Turnip Yellows Virus Infectious Clones: A New Way to Monitor Plant Virus Infection

Boissinot

Ducousso

Brault

et al. 2022

IJMS

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We used the NanoLuc luciferase bioluminescent reporter system to detect turnip yellows virus (TuYV) in infected plants. For this, TuYV was genetically tagged by replacing the C-terminal part of the RT protein with full-length NanoLuc (TuYV-NL) or with the N-terminal domain of split NanoLuc (TuYV-N65-NL). Wild-type and recombinant viruses were agro-infiltrated in Nicotiana benthamiana, Montia perfoliata, and Arabidopsis thaliana. ELISA confirmed systemic infection and similar accumulation of the recombinant viruses in N. benthamiana and M. perfoliata but reduced systemic infection and lower accumulation in A. thaliana. RT-PCR analysis indicated that the recombinant sequences were stable in N. benthamiana and M. perfoliata but not in A. thaliana. Bioluminescence imaging detected TuYV-NL in inoculated and systemically infected leaves. For the detection of split NanoLuc, we constructed transgenic N. benthamiana plants expressing the C-terminal domain of split NanoLuc. Bioluminescence imaging of these plants after agro-infiltration with TuYV-N65-NL allowed the detection of the virus in systemically infected leaves. Taken together, our results show that NanoLuc luciferase can be used to monitor infection with TuYV.

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Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors

Cited by 7 publications

References 24 publications

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release

Advances in experimental systems to study hepatitis C virus in vitro and in vivo

Bioluminescence Production by Turnip Yellows Virus Infectious Clones: A New Way to Monitor Plant Virus Infection

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